Method for improving the solubility of vegetable proteins

ABSTRACT

The present invention relates to a method for improving the solubility of vegetable proteins. More specifically, the invention relates to methods for the solubilization of proteins in vegetable protein sources, which methods comprise treating the vegetable protein source with an efficient amount of one or more phytase enzymes, and treating the vegetable protein source with an efficient amount of one or more proteolytic enzymes. In another aspect, the invention provides animal feed additives comprising a phytase and one or more proteolytic enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK95/00166 filed Apr. 20, 1995, which is incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a method for improving the solubilityof vegetable proteins. More specifically, the invention relates tomethods for the solubilization of proteins in vegetable protein sources,which methods comprise treating the vegetable protein source with anefficient amount of one or more phytase enzymes, and treating thevegetable protein source with an efficient amount of one or moreproteolytic enzymes. In another aspect the invention provides animalfeed additives comprising a phytase and one or more proteolytic enzymes.

BACKGROUND ART

Protein is an essential nutritional factor for a.o. mammals and layers.Most livestock and many human beings get the necessary proteins fromvegetable protein sources. Important vegetable protein sources are e.g.cereals, legumes and oilseed crops.

Hydrolyzed vegetable proteins, such as soy protein hydrolysates, findapplication as nutrients, e.g. as nutritional additives to foods andbeverages. Hydrolyzed proteins are absorbed more easily thanunhydrolysed protein, whey protein hydrolysates are considered havingthe highest nutritional value. Several methods for preparing proteinhydrolysates are known and described in the literature, cf. e.g. U.S.Pat. No. 4,324,805 and WO 92/15696.

Essentially all food and feed substances originating from plants containphytate and phytic acid as a storage phosphorus source [cf. review in E.Graf (ed.), Phytic Acid, Chemistry and Applications, Minneapolis,U.S.A., 1986]. About 75-78% of the phosphorus in cereals is bound asphytic acid. Phytate comprises 1-3% of all nuts, cereals, legumes, oilseeds, spores and pollen. Complex salts of phytic acid are termedphytin.

Phytic acid chelates minerals such as calcium, zinc, magnesium, andiron, thereby decreasing the bio-availability of nutritionally importantminerals, and is generally considered an anti-nutritional factor. Invitro studies indicate that phytic acid inhibits the peptic digestion ofsome animal proteins, whereas the trypsin digestion was unaffected [cf.Knuckles et al., Journal of Food Science. 1989 54 1348-1350].

Phytases are enzymes which catalyze the conversion of phytate toinositol and inorganic phosphorus. Phytases have been obtained from e.g.Bacillus, Pseudomonas, Saccharomyces and Aspergillus.

In U.S. Pat. No. 3,297,548 it has been suggested to add microbialphytase to feedstuffs of monogastric animals in order to avoidsupplementing the feed with inorganic phosphorus.

It has also been conceived that phytases may be used in the processingof soy. Thus EP-A-0 420 358 reports that soybean meal contains highlevels of the anti-nutritional factor phytate which renders this proteinsource unsuitable for application in baby food, as well as in feed forfish, calves and other non-ruminants, since the phytate chelatesessential minerals present therein.

In summary it has previously been suggested to use phytase enzymeseither for exploiting the phosphorus bound in the phytate/phytic acidpresent in vegetable protein sources, or for exploiting thenutritionally important minerals bound in phytic acid complexes.

However, there is a need for improving the availability of the proteinspresent in vegetable sources, e.g. cereals, legumes and oilseed crops,since an increased availability leads to higher yields of proteinhydrolyzing processes as well as nutritional benefits, i.e. improvedprotein utilization.

SUMMARY OF THE INVENTION

It has now been found that the solubility of proteins present invegetable sources may be increased by treating the vegetable source withan efficient amount of a phytase enzyme. By addition of a phytase duringa proteolytic process, higher degrees of hydrolysis and improved proteinsolubility are obtained, and yields are improved.

Accordingly, the present invention provides a method for solubilizingproteins in a vegetable protein source, which method comprises treatingthe vegetable protein source with an efficient amount of one or morephytase enzymes, and treating the vegetable protein source with anefficient amount of one or more proteolytic enzymes.

In another aspect, the invention provides an animal feed additivecomprising a phytase and a proteolytic enzyme.

DETAILED DISCLOSURE OF THE INVENTION

Solubilization of Vegetable Proteins

The present invention provides a method for the solubilization ofproteins in a vegetable protein source, thereby obtaining a proteinhydrolysate. The method comprises the steps of

(a) treating the vegetable protein source with an efficient amount ofone or more phytase enzymes; and

(b) treating the vegetable protein source with an efficient amount ofone or more proteolytic enzymes.

Compared to known method for obtaining protein hydrolysates, the presentmethod improves the availability of the proteins, thereby leading toincreased extraction, higher yields and improved protein utilization.

The vegetable protein source subjected to the method of the inventionmay be any proteinaceous vegetable, in particular a legume, a cereal, acomposite plant or a crucifera. Preferably, the legume is selected fromthe group consisting of soy bean, faba bean, pea, and lupine (Lupinusalbus), more preferably the legume is soy beans. Preferably, the cerealis selected from the group consisting of wheat, corn, barley, rye, oat,rice, sorghum, sesame (Sesamum indicum), and millet (Panicum miliaceum).An example of a useful composite plant is sunflower (Helianthus), and anexample of a useful crucifera is rape (rape seed, e.g. Brassica napus).

The vegetable protein source is preferably soy bean.

The proteinaceous vegetable subjected to the method of the invention maybe provided in any form, including "processed forms" wherein the proteincontent of the dry matter is increased before the protein subjected tothe method according to the invention. For instance, soy protein rawmaterial may obtained as soy beans, defatted soy flakes, soy meal, soyconcentrate or soy isolate. Such proteinaceous raw material typicallycontains from about 42% to about 95% protein.

The protein source may be pre-treated in any conventional manner priorto being subjected to the method of the present invention. In particularthe protein source may be dried and/or grounded, i.e. processed intomeal or flakes (such as soy bean meal or flakes).

The two steps, (a) and (b) of the method of the invention, may becarried out as consecutive steps, or the two steps may be carried outsimultaneously.

Also, the vegetable protein source may be treated with one enzymepreparation comprising one or more phytase enzymes and one or moreproteolytic enzymes, or the vegetable protein source may be treated withtwo or more enzyme comprising a one or more phytase enzymes and/or oneor more proteolytic enzymes.

It is at present contemplated that the method of the invention ispreferably carried out at a pH of the suspension of from about 3 toabout 9, more preferably of from about pH 4 to about pH 7.

It is also contemplated that the method of the invention is preferablycarried out at a temperature of from about 10° C. to about 70° C., morepreferably of from about 35° C. to about 65° C.

In a more specific embodiment, the method comprises the subsequent stepsof

(i) suspending a vegetable protein source in water;

(ii) proteolytically hydrolysing the protein by subjecting thesuspension to enzymatic treatment with one or more phytase enzymes andone or more proteolytic enzyme;

(iii) inactivating the enzymes; and optionally

(iv) separating the hydrolysed protein from the suspension.

Inactivation of the enzyme may be carried out by conventional methodsfor inactivating enzymes, e.g. by heat treatment, i.e. elevating thetemperature of the hydrolysis suspension or mixture to a temperaturewhich denatures the enzymes, typically to a temperature of above 85° C.

The separating step may be carried out by conventional methods forseparating hydrolysed protein from suspensions, e.g. by centrifugationor membrane filtrating the suspension containing the proteinhydrolysate.

In general terms, the method of the invention may be carried out similarto conventional methods for obtaining protein hydrolysates, e.g. asdescribed in U.S. Pat. No. 4,324,805, U.S. Pat. No. 4,100,024 and WO92/15696, which publications are hereby incorporated by reference.

Phytase Enzymes

The method of the invention for the solubilization of proteins in avegetable protein source comprises treating the vegetable protein sourcewith an efficient amount of one or more phytase enzymes.

In the context of this invention, a phytase enzyme is an enzyme whichcatalyzes the removal of inorganic phosphorous from various myoinositolphosphates. Phytase enzymes are preferably derived from a microbialsource such as bacteria, fungi and yeasts, but may also be of vegetableorigin.

In a preferred embodiment, the phytase enzyme is derived from a fungalstrain, in particular a strain of Aspergillus, e.g Aspergillus niger,Aspergillus oryzae, Aspergillus ficuum, Aspergillus awamori, Aspergillusnidulans and Aspergillus terreus. Most preferred is a phytase enzymederived from a strain of Aspergillus niger or a strain of Aspergllusoryzae.

In another preferred embodiment, the phytase enzyme is derived from abacterial strain, in particular a strain of Bacillus or a strain ofPseudomonas. Preferably the phytase enzyme is derived from a strain ofBacillus subtilis.

In yet another preferred embodiment, the phytase enzyme is derived froma yeast, in particular a strain of Kluveromyces or a strain ofSaccharomyces. Preferably the phytase enzyme is derived from a strain ofSaccharomyces cerevisiae.

In the context of this invention "an enzyme derived from" encompasses anenzyme naturally produced by the particular strain, either recoveredfrom that strain or encoded by a DNA sequence isolated from this strainand produced in a host organism transformed with said DNA sequence.

The phytase enzyme may be derived from the microorganism in question byuse of any suitable technique. In particular, the phytase enzyme may beobtained by fermentation of a phytase producing microorganism in asuitable nutrient medium, followed by isolation of the enzyme by methodsknown in the art.

The broth or medium used for culturing may be any conventional mediumsuitable for growing the host cell in question, and may be composedaccording to the principles of the prior art. The medium preferablycontain carbon and nitrogen sources and other inorganic salts. Suitablemedia, e.g. minimal or complex media, are available from commercialsuppliers, or may be prepared according to published receipts, e.g. theAmerican Type Culture Collection (ATCC) Catalogue of strains.

After cultivation, the phytase enzyme is recovered by conventionalmethod for isolation and purification proteins from a culture broth.Well-known purification procedures include separating the cells from themedium by centrifugation or filtration, precipitating proteinaceouscomponents of the medium by means of a salt such as ammonium sulphate,and chromatographic methods such as e.g. ion exchange chromatography,gel filtration chromatography, affinity chromatography, etc.

Alternatively, the phytase enzyme is preferably produced in largerquantities using recombinant DNA techniques, e.g. as described inEP-A1-0 420 358, which publication is hereby incorporated by reference.

Preferably, a fungus of the species Aspergillus which has beentransformed with the phytase-encoding gene obtained from the speciesAspergillus ficuum or Aspergillus niger, is cultured under conditionsconducive to the expression of the phytase-encoding gene as described inEP-A1-0 420 358.

The phytase-containing fermentation broth is preferably treated by meansof both filtration and ultra-filtration prior to being used in the(formulation) of the present invention.

It is at present contemplated that an amount of phytase efficient forimproving the solubility of the vegetable protein corresponds to of fromabout 2 to about 50000 FYT (as defined below) per kg of vegetableprotein source, preferably of from about 50 to about 30000 FYT per kg ofvegetable protein source, most preferred of from about 100 to about10000 FYT per kg of vegetable protein source.

Proteolytic Enzymes

The method of the invention for the solubilization of proteins in avegetable protein source also comprises treating the vegetable proteinsource with an efficient amount of one or more proteolytic enzymes.

The proteolytic enzyme may be a microbial enzyme, preferably a proteasederived from a bacterial or a fungal strain, or the proteolytic enzymemay be trypsin or pepsin. In a preferred embodiment, the proteolyticenzyme is a bacterial protease derived from a strain of Bacillus,preferably a strain of Bacillus subtilis or a strain of Bacilluslicheniformis. Commercially available Bacillus proteases are Alcalase™and Neutrase™ (Novo Nordisk A/S, Denmark). In another preferredembodiment, the proteolytic enzyme is a fungal protease derived from astrain of Aspergillus, preferably a strain of Aspergillus aculeatus, astrain of Aspergillus niger, a strain of Aspergillus oryzae. Acommercially available Aspergillus protease is Flavourzyme™ (NovoNordisk A/S, Denmark).

It is at present contemplated that the amount of proteolytic enzymeefficient for improving the solubility of the vegetable proteincorresponds to of from about 0.0001 to about 0.5 AU (as defined below)per kg of vegetable protein source, preferably of from about 0.001 toabout 0.05 AU per kg of vegetable protein source, more preferred of fromabout 0.005 to about 0.03 AU per kg of vegetable protein source.

Additional Enzymes

As described above, the present invention provides a method for thesolubilization of proteins in a vegetable protein source, which methodcomprises the steps of

(a) treating the vegetable protein source with an efficient amount ofone or more phytase enzymes; and

(b) treating the vegetable protein source with an efficient amount ofone or more proteolytic enzymes.

The two steps, (a) and (b) of the method of the invention, may becarried out as consecutive steps, or the two steps may be carried outsimultaneously.

In a preferred embodiment, the method of the invention further comprisesthe step of

(c) treating the vegetable protein source with an efficient amount ofone or more enzymes selected from the group consisting of lipolyticenzymes and glucosidase enzymes.

The three steps, (a), (b) and (c) of the method of the invention, may becarried out as consecutive steps, or the three steps may be carried outsimultaneously. Or the two steps, (a) and (b) of the method of theinvention, may be carried out simultaneous, followed by step (c).

The lipolytic enzyme of step (c) may be any triacylglycerol lipase (EC3.1.1.3).

The glycosidase enzyme of step (c) may be any glycosidase enzyme (EC3.2, also known as carbohydrases). Preferably, the glycosidase enzyme isan amylase, in particular an α-amylase or a β-amylase, a cellulase, inparticular an endo-1,4-β-glucanase (EC 3.2.1.4) or anendo-1,3-β-glucanase (3.2.1.6), a xylanase, in particular anendo-1,4-β-glucanase (EC 3.2.1.8) or a xylan-endo-1,3-β-xylosidase (EC3.2.1.32), an α-galactosidase (EC 3.2.1.22), a polygalacturonase (EC3.2.1.15, also known as pectinases), a cellulose-1,4-β-cellobiosidase(EC 3.2.1.91, also known as cellobiohydrolases), an endoglucanase, inparticular an endo-1,6-σ-glucanase (EC 3.2.1.75), anendo-1,2-β-glucanase (EC 3.2.1.71), an endo-1,3-β-glucanase (EC3.2.1.39) or an endo-1,3-α-glucanase (EC 3.2.1.59).

In a more preferred embodiment, the glucosidase enzyme is apolygalacturonase (EC 3.2.1.15), a cellulose-1 ,4-β-cellobiosidase (EC3.2.1.91), or an endoglucanase, preferably an endo-1,6-β-glucanase (EC3.2.1.75), an endo-1,2-β-glucanase (EC 3.2.1.71), anendo-1,3-β-glucanase (EC 3.2.1.39) or an endo-1,3-α-glucanase (EC3.2.1.59).

Industrial Applications

The method of the invention for the solubilization of proteins in avegetable protein source, may find application in various industries. Inparticular the method of the invention find application for thepreparation of protein hydrolysates useful as nutrients, e.g. asnutritional additives to foods and beverages.

Therefore, in another aspect, the present invention also provides aprotein hydrolysate obtained from a vegetable protein source by themethod of the invention.

The protein hydrolysate of the invention may be added to foods orbeverages, thereby increasing the nutritional value.

In particular the protein hydrolysate of the invention may be added toanimal feed, e.g. feedstuffs for monogastric animals.

Animal Feed Additives

The present invention provides an animal feed additive comprising one ormore phytase enzymes and one or more proteolytic enzymes.

When added to animal feed, the combination of one or more phytaseenzymes and one or more proteolytic enzymes improves the availability ofthe proteins present in animal feed of vegetable origin, thereby leadingto increased extraction of the vegetable proteins, higher protein yieldsand improved protein utilization. Thereby, the nitrogen digestibilityand the nutritive value of the fodder becomes increased, and the growthrate and/or feed conversion ratio (i.e. the weight of ingested feedrelative to weight gain) of the animal is improved.

In the context of this invention, an animal feed additive is an enzymepreparation comprising one or more phytase enzymes and one or moreproteolytic enzymes and suitable carriers and/or excipients, and whichenzyme preparation is provided in a form that is suitable for beingadded to animal feed. The animal feed additive of the invention may beprepared in accordance with methods known in the art and may be in theform of a dry or a liquid preparation. The enzyme to be included in thepreparation, may optionally be stabilized in accordance with methodsknown in the art.

In a specific embodiment the animal feed additive of the invention is agranulated enzyme product which may readily be mixed with feedcomponents, or more preferably, form a component of a pre-mix. Thegranulated enzyme product may be coated or uncoated. The particle sizeof the enzyme granulates preferably is compatible with that of feed andpre-mix components. This provides a safe and convenient mean ofincorporating the enzymes into feeds.

In another specific embodiment, the animal feed additive of theinvention is a stabilized liquid composition, which may be an aqueous oroil-based slurry.

In yet another specific embodiment, one or more of the enzymes areapplied before or after pelleting or extrusion of the feed.

The animal feed additive of the invention may exert its effect either invitro (by modifying components of the feed) or in vivo. The feedadditive of the invention is particularly suited for addition to animalfeed compositions containing high amounts of proteinaceous vegetables,in particular legumes, cereals, composite plants or crucifera.Preferably, the legume is soy bean, faba bean, pea, and/or lupine(Lupinus albus). Most preferably the legume is soy beans. Preferably,the cereal is wheat, corn, barley, rye, oat, rice, sorghum, sesame(Sesamum indicum), and millet (Panicum miliaceum). An example of acomposite plant is sunflower (Helianthus), and an example of a usefulcrucifera is rape (rape seed, e.g. Brassica napus).

In another preferred embodiment, the invention provides an animal feedadditive which comprises one or more additional enzymes selected fromthe group consisting of lipolytic enzymes and glucosidase enzymes.

The lipolytic enzyme may be any triacylglycerol lipase (EC 3.1.1.3).

The glycosidase enzyme may be any glycosidase enzyme (EC 3.2, also knownas carbohydrases). Preferably, the glycosidase enzyme is an amylase, inparticular an α-amylase or a β-amylase, a cellulase, in particular anendo-1,4-β-glucanase (EC 3.2.1.4) or an endo-1,3-β-glucanase (3.2.1.6),a xylanase, in particular an endo-1,4-β-glucanase (EC 3.2.1.8) or axylan-endo-1,3-β-xylosidase (EC 3.2.1.32), an α-galactosidase (EC3.2.1.22), a polygalacturonase (EC 3.2.1.15, also known as pectinases),a cellulose-1,4-β-cellobiosidase (EC 3.2.1.91, also known ascellobiohydrolases), an endoglucanase, in particular anendo-1,6-σ-glucanase (EC 3.2.1.75), an endo-1,2-β-glucanase (EC3.2.1.71), an endo-1,3-β-glucanase (EC 3.2.1.39) or anendo-1,3-α-glucanase (EC 3.2.1.59).

In a more preferred embodiment, the glucosidase enzyme is apolygalacturonase (EC 3.2.1.15), a cellulose-1,4-β-cellobiosidase (EC3.2.1.91), or an endoglucanase, preferably an endo-1,6-β-glucanase (EC3.2.1.75), an endo-1,2-β-glucanase (EC 3.2.1.71), anendo-1,3-β-glucanase (EC 3.2.1.39) or an endo-1,3-α-glucanase (EC3.2.1.59).

It is at present contemplated that the pH of the animal feed additiveshould be in the range of from about pH 2 to about pH 8.

It is at present contemplated that the amount of phytase activity in theanimal feed additive should be in the range of from about 200 to about50000 FYT (as defined below) per gram of the total composition,preferably in the range of from about 500 to about 10000 FYT per gram ofthe total composition, most preferred in the range of from about 2000 toabout 6000 FYT per gram of the total composition.

Preferably, the amount of additive added to the animal feed should besufficient to provide a feed composition containing at least 50 FYT perkg feed, more preferably, between about 100 and about 2000 FYT per kgfeed.

It is to be understood that the actual amount of Phytase Units whichshould be added to the feed in order to enhance the nutritive value,i.e. the nitrogen digestibility, will depend on the composition of thefeed itself. Feedstuffs containing high amounts of phytic acid willgenerally require the addition of higher amounts of phytase activity.The necessary amount of phytase may easily by determined by the skilledperson.

Phytase Activity (FYT)

The phytase activity may be determined using sodium phytate assubstrate. When subjected to the action of phytase, inorganicphosphorous is liberated from sodium phytate. In a ferrous/molybdenumcontaining reagent, phosphorous (PO₄) forms a complex, which can bemonitored spectrophotometrically at 750 nm.

One Phytase Unit (FYT)is defined as the amount of enzyme which understandard conditions (i.e. at pH 5.5, 37° C., a substrate concentrationof 5.0 mM sodium phytate, and a reaction time of 30 minutes) liberates 1μmol of phosphate per minute.

A standard operating procedure EAL-SM-0403.01 describing this analyticalmethod in more detail is available upon request to Novo Nordisk A/S,Denmark, which publication is hereby included by reference.

Protease Activity (AU)

The proteolytic activity may be determined with denatured hemoglobin assubstrate. In the Anson-Hemoglobin method for the determination ofproteolytic activity denatured hemoglobin is digested, and theundigested hemoglobin is precipitated with trichloroacetic acid (TCA).The amount of TCA soluble product is determined with phenol reagent,which gives a blue color with tyrosine and tryptophan.

One Anson Unit (AU) is defined as the amount of enzyme which understandard conditions (i.e. 25° C., pH 7.5 and 10 min. reaction time)digests hemoglobin at an initial rate such that there is liberated perminute an amount of TCA soluble product which gives the same color withphenol reagent as one milliequivalent of tyrosine.

A folder AF 4/5 describing the analytical method in more detail isavailable upon request to Novo Nordisk A/S, Denmark, which folder ishereby included by reference.

EXAMPLES

The invention is further illustrated with reference to the followingexamples which are not intended to be in any way limiting to the scopeof the invention as claimed.

Example 1

Soy Protein Hydrolysis

Soy protein concentrate (Unico 75, Loders Crooklaan, NL) was suspendedin deionized water at 50° C.

The mixture was heat treated at 85° C. for 3 minutes and cooled to 55°C., the temperature of hydrolysis. pH was adjusted to 8.5 using 4 NNaOH.

The mixture was divided into three vessels for three comparativeexperiments:

1) Hydrolysing with:

Alcalase™ 2.4 L, dosage 2 w/w % of protein content.

Neutrase™ 0.5 L, dosage 1 w/w % of protein content.

Phytase Novo™ (an Aspergillus phytase, 6900 FYT/g), dosage 1 mg/g soyconcentrate. pH adjusted to 4.5 with HCl. Phytase is added when pH<6.6.

2) Hydrolysing with:

Alcalase™ 2.4 L, dosage 2 w/w % of protein content.

Neutrase™ 0.5 L, dosage 1 w/w % of protein content.

3) pH was adjusted to 6.2 using HCl. Phytase Novo™ (an Aspergillusphytase, 6900 FYT/g), dosage 1 mg/g soy concentrate.

The three hydrolyses were followed by monitoring the degree ofhydrolysis and the osmolality. The results are presented in Tables 1-2,below.

                  TABLE 1                                                         ______________________________________                                        Osmolality, mOsm/kg.                                                          Time (minutes)                                                                            Exp. 1)     Exp. 2) Exp. 3)                                       ______________________________________                                        0           103         102     103                                           15          200         173     145                                           35          211         183     147                                           65          232         201     139                                           125         241         208     142                                           185         250         221     140                                           ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Degree of Hydrolysis, % DH                                                    Time (minutes)                                                                            Exp. 1)     Exp. 2) Exp. 3)                                       ______________________________________                                        20          12.2        11.9    -1.9                                          40          13.9        12.6    -1.6                                          70          13.2        12.8    -2.0                                          130         17.3        15.0    -2.6                                          190         19.5        18.2    -2.4                                          ______________________________________                                    

Both measurements of the degree of hydrolysis (%DH) show an increaseddegradation of the substrate. Experiment 1 yielded more solubilizedprotein (78.8%) when compared to Experiment 2 (77.3%).

Example 2

In Vitro N-digestibility of Soy Bean Meal

Two samples were prepared:

Sample A: 1 g of soybean meal

Sample B: 1 g of soybean meal with addition of 0.032 g Phytase Novo™ (anAspergillus phytase, 5000 FYT/g)

Each sample was incubated with pepsin at pH 2.4 for 2 hours followed byincubation with pancreatin at pH 6.8 for 16 hours. Suspended but notdigested protein was precipitated with sulfosalicylic acid. Theundigested protein (as well as other undigested components) wascollected by filtration and drying. The dry matter of the filter cakeand of the sample was determined, and the amount of nitrogen (N) wasdetermined by the Kjeldahl method.

The N digestibility was calculated as follows: ##EQU1##

The protein digestibility was calculated by multiplying the nitrogen (N)digestibility by 6.25.

The following protein digestibility results were obtained:

Sample A (without phytase): 88.4%

Sample B (with phytase): 89.9%

Example 3

Effect of Phytase on Apparent Nitrogen Digestibility and NitrogenUtilization in Pigs

Balance trials on pigs (weight 46-52 kg) on a barley-wheat-soy diet.

Diet composition:

Barley 50.8%

Wheat 20.0%

Soy 24.0%

Animal fat 2.0%

Melasse 1.0%

Minerals, vitamins and amino acids 2.2%

No addition of inorganic phosphate to the diet. The diet was pelletizedat a temperature of above 60° C.

Balance trial was conducted with an adaption period of 5 days and a 7days collection period.

12 pigs were divided into two groups. The first group was fed the dietand the second group was fed the same diet but with addition of 20.3g/100 kg feed of Phytase Novo™ (an Aspergillus phytase, 7370 FYT/g).

The content of nitrogen in the diet, faeces and urine was measured andthe apparent nitrogen digestibility and nitrogen utilization wascalculated.

The results are presented in Table 3, below.

                  TABLE 3                                                         ______________________________________                                        Nitrogen Digestibility and Nitrogen Utilization                               Nitrogen (g/day)                                                                        Diet without Phytase                                                                        Diet with Phytase                                                                         S.E.M.                                    ______________________________________                                        Consumed  44.5          44.5                                                  Faeces    6.8           6.2         0.1*                                      Urine     20.3          19.5        0.8 NS                                    Deposit   17.4          18.7        0.6 NS                                    Digested  37.6          38.3        0.1*                                      Digested, %                                                                             84.7          86.1        0.3*                                      ______________________________________                                         *p ≦ 0.01                                                              NS (not significant): p > 0, 05                                               S.E.M.: standard error mean                                              

We claim:
 1. An animal feed additive comprising one or more phytases andone or more bacterial proteolytic enzymes.
 2. The animal feed additiveof claim 1, further comprising one or more additional enzymes selectedfrom the group consisting of lipolytic enzymes and glucosidase enzymes.3. The animal feed additive of claim 2, in which the glucosidase enzymeis selected from the group consisting of a carbohydrase, amylase,cellulase, xylanase, α-galactosidase, polygalacturonase, andcellobiohydrolase.
 4. The animal feed additive of claim 2, in which theglucosidase enzyme is a polygalacturonase,cellulose-1,4-β-cellobiosidase, or endoglucanase.
 5. The animal feedadditive of claim 1, wherein the phytase is a vegetable phytase, fungalphytase, bacterial phytase, or phytase obtained from a yeast.
 6. Theanimal feed additive of claim 5, wherein the phytase is a fungal phytasederived from a strain of Aspergillus.
 7. The animal feed additive ofclaim 5, wherein the phytase is a bacterial phytase derived from astrain of Bacillus or Pseudomonas.
 8. The animal feed additive of claim5, wherein the phytase is derived from Kluveromyces or Saccharomnyces.9. The animal feed additive of claim 1, wherein the protease is derivedfrom a strain of Bacillus.
 10. An animal feed comprising one or morefeed components, one or more phytases and one or more bacterialproteolytic enzymes.
 11. The animal feed of claim 10, further comprisingone or more additional enzymes selected from the group consisting oflipolytic enzymes and glucosidase enzymes.
 12. The animal feed of claim11, in which the glucosidase enzyme is selected from the groupconsisting of a carbohydrase, amylase, cellulase, xylanase,α-galactosidase, polygalacturonase, and cellobiohydrolase.
 13. Theanimal feed of claim 11, in which the glucosidase enzyme is apolygalacturonase, cellulose-1,4-β-cellobiosidase, or endoglucanase. 14.An animal feed comprising a protein hydrolyzate obtained by treating avegetable protein source with one or more phytases and one or morebacterial proteolytic enzymes.
 15. A method solubilizing proteins in avegetable protein source, which method comprises the steps of(a)treating the vegetable protein source with an efficient amount of one ormore phytase enzymes; (b) treating the vegetable protein source with anefficient amount of one or more bacterial proteolytic enzymes; and (c)treating the vegetable protein source with an efficient amount of one ormore lipolytic enzymes and/or one or more glucosidase enzymes.
 16. Themethod of claim 15, in which the glucosidase enzyme is selected from thegroup consisting of a carbohydrase, amylase, cellulase, xylanase,α-galactosidase, polygalacturonase, and cellobiohydrolase.
 17. Themethod of claim 16, in which the glucosidase enzyme is apolygalacturonase, cellulose-1,4-β-cellobiosidase, or endoglucanase. 18.The method of claim 15, wherein steps (a), (b) and (c) are carried outsimultaneously.
 19. The method of claim 15, wherein the phytase is avegetable phytase, fungal phytase, bacterial phytase or phytase derivedfrom a yeast.
 20. The method of claim 19, wherein the phytase is afungal phytase derived from a strain of Aspergillus.
 21. The method ofclaim 19, wherein the phytase is a bacterial phytase derived from astrain of Bacillus or Pseudomonas.
 22. The method of claim 19, whereinthe phytase is derived from Kluveromyces or Saccharomyces.
 23. Themethod of claim 15, wherein the protease is derived from a strain ofBacillus.
 24. The method of claim 15, wherein the vegetable proteinsource is a legume, composite plant, crucifera, cereal, or a mixturethereof.
 25. The method of claim 15, wherein the vegetable proteinsource is soy bean.
 26. The method of claim 15, wherein the enzymatictreatment is carried out at a pH of from 3 to
 9. 27. The method of claim15, wherein the enzymatic treatment is carried out at a temperature offrom about 10 to about 70° C.
 28. A method of producing a proteinhydrolyzate, comprising(a) treating a vegetable protein source with anefficient amount of one or more phytase enzymes; (b) treating thevegetable protein source with an efficient amount of one or morebacterial proteolytic enzymes; and (c) treating the vegetable proteinsource with an efficient amount of one or more lipolytic enzymes and/orone or more glucosidase enzymes.
 29. The method of claim 28, in whichthe glucosidase enzyme is selected from the group consisting of acarbohydrase, amylase, cellulase, xylanase, α-galactosidase,polygalacturonase, and cellobiohydrolase.
 30. The method of claim 29, inwhich the glucosidase enzyme is a polygalacturonase,cellulose-1,4-β-cellobiosidase, or endoglucanase.
 31. Amethod ofincreasing nitrogen digestibility and the nutritive value of food orimproving the growth rate and feed conversion ratio of an animal,comprising feeding the animal with an animal feed of claim 10.